Project 2: Structure and function of poxvirus immune evasion domains. In this project we will explore the functional and mechanistic attributes of a large family of sequence diverse proteins encoded by the zoonotic orthopoxvirus cowpox. We hypothesize that ten proteins In cowpox share a similar structural scaffolding that we have termed the poxvirus immune evasion (PIE) domain. Certain PIE proteins are known to sabotage host Immune surveillance and effector functions, and our goal Is to systematically determine the ligands and receptors for all ten PIE proteins encoded by the Brighton Red strain of cowpox. These efforts will be complemented by crystallographic investigations and quantitative binding assays to determine PIE protein structure and specificity, and structure-based engineering to develop variants with altered ligand/receptor Interactions. We will also undertake extensive In vitro functional studies of the PlEs, evaluating their mechanisms of action In the context of well-established Immunological assays. Our investigations will also be considered In the broader context of the battlefield of viral infection and host defense. In close collaboration with the Yokoyama and Fruh labs we will evaluate the pathogenesis of cowpox with either selective PIE proteins knocked out or their ligand/receptor binding determinants altered. The proposed experiments will result In a significantly improved functional annotation of cowpox and yield important clues about host-pathogen Interactions that are likely to benefit both the scientific and clinical communities.